REVIEW

Australian Dental Journal 1999;44:(3):147-156

Current concepts in oral cancer

Philip B. Sugerman, BDS, PhD, FRACDS, FDSRCS, FFOP RCPA* Neil W. Savage, MDSc, PhD, FFOP RCPA, FICD

Abstract Over 750 new intra-oral squamous cell carcinomas are registered in Australia each year. In this article, the authors review the epidemiology, aetiology, genetics and spread of intra-oral squamous cell carcinoma. The mechanisms of field cancerization are discussed. The prevention of intra-oral squamous cell carcinoma is highlighted and future treatments are presented.
Key words: Oral cancer, epidemiology, aetiology, genetics, spread, prevention, therapy. (Received for publication February 1999. Revised June 1999. Accepted June 1999.)

Introduction Intra-oral squamous cell carcinoma accounts for approximately one per cent of all cancers and one per cent of all cancer-related deaths in men and women in Australia. It is estimated that 75 per cent of all intra-oral squamous cell carcinomas in Australia are attributable to smoking and alcohol. Therefore, patient awareness of the risks of smoking and alcohol may help prevent many intra-oral squamous cell carcinomas. Cancer in Australia The anatomical location of malignancies is coded by the World Health Organization (WHO).1 There were 75 498 new cancers (excluding non-melanocytic skin cancer) registered in Australia in 1994. Prostate cancer was the most common followed by colorectal cancer, breast cancer, lung cancer and melanoma (Table 1). Of the 42 619 cancers registered in males in Australia in 1994, the most common was prostate cancer. Of the 32 879 cancers registered in females in Australia in 1994, the most common was breast cancer (Table 2). Lung cancer in males and breast cancer in females were the most common cancers causing death in Australia in 1994 (Table 3).2
*CJ Martin Postdoctoral Fellow and Registrar in Oral Medicine and Pathology, Department of Dentistr y, The University of Queensland. Reader in Oral Medicine and Pathology, Department of Dentistr y, The University of Queensland.
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Oral cancer in Australia The 1994 Australian oral cancer incidence and mortality data are presented in Table 4.There were 1906 new oral cancers (all sites) registered in Australia in 1994; 1299 in males and 607 in females. As a comparison, there were 1121 new cancers of the cervix uteri (ICD9-180) registered in Australia in 1994.The lip is by far the most common site for cancer in the oral region (Table 4).The incidence of oral cancer in Australia is relatively stable over time. The authors define intra-oral cancer as cancer affecting the oral tissues excluding lip cancer (ICD9140) and major salivary gland cancer (ICD9-142). Intra-oral cancer includes tongue (ICD9-141), gum (ICD9-143), floor of mouth (ICD9-144) and other mouth (ICD9-145). Using this definition, there were 804 new intra-oral cancers registered in Australia in 1994; 515 in males and 289 in females. The tongue is the most common site for intra-oral cancer in Australia and the majority of intra-oral cancers (all sites) are in men (Table 4). Conservative estimates suggest that 95 per cent of intra-oral cancers are squamous cell carcinomas (SCCs). Hence, there were approximately 764 new intra-oral SCCs registered in Australia in 1994; 489 in males and 275 in females. Intra-oral SCC therefore accounts for approximately one per cent of all cancers in men and women in Australia. Deaths resulting from intra-oral SCC (288 in 1994) account for approximately one per cent of all cancer-related deaths in Australia.2 The mortality to incidence (M:I) ratio for oral cancers at various sites is shown in Table 4. The data give no

Table 1. The most common cancers registered in Australia in 19942*

Rank 1 2 3 4 5 Cancer prostate (ICD9-185) colorectal (ICD9-153/154) breast (ICD9-174/175) lung (ICD9-162) melanoma (ICD9-172) Number of cases 12 787 10 016 9 764 7 306 6 776

*The anatomical location of malignancies is coded by the World Health Organization.1

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Table 2. The most common male and female cancers registered in Australia in 1994 (number of cases)2*
Rank 1 2 3 4 Males prostate (ICD9-185) colorectal (ICD9-153/154) lung (ICD9-162) melanoma (ICD9-172) 12 787 5 433 5 196 3 695 Females breast (ICD9-174) colorectal (ICD9-153/154) melanoma (ICD9-172) lung (ICD9-162) 9 694 4 583 3 801 2 110

*The anatomical location of malignancies is coded by the World Health Organization. 1

Table 3. The most common male and female cancers causing death in Australia in 1994 (number of deaths)2*
Rank 1 2 3 Males lung (ICD9-162) prostate (ICD9-185) colorectal (ICD9-153/154) 4 833 2 613 2 501 Females breast (ICD9-174) colorectal (ICD9-153/154) lung (ICD9-162) 2 669 2 126 1 901

*The anatomical location of malignancies is coded by the World Health Organization. 1

indication of the behaviour of individual tumours, although they suggest that tongue, major salivary gland and floor of mouth cancers have the greatest lethality, whereas lip cancer has relatively low lethality. In the USA,an estimated 29 800 new cases of oral cancer are expected to be diagnosed in 1999 and approximately 8100 people will die of the disease. Oral cancer accounts for about three per cent of cancers in men and two per cent of cancers in women. More than 90 per cent of oral cancers occur in patients over the age of 45.The incidence increases steadily with age until 65, when the rate levels off. Over the last 22 years in the USA, there have been slight decreases in oral cancer incidence and mortality rates.Worldwide, oral SCC is the sixth most common malignancy. Although oral SCC accounts for less than five per cent of malignant tumours in developed countries, in parts of India and South East Asia oral SCC is the most common malignancy, accounting for up to 50 per cent of malignant tumours in these regions. The significance of oral cancer involves its mortality and the disfigurement and dysfunction associated with treatment. The rate of curability of cancers of the lip and oral cavity varies depending on the stage and the site. Most lip cancers and small cancers of the retromolar trigone, hard palate and upper gingiva are highly curable by surgery or radiation therapy. Small cancers of the anterior tongue, the floor of the mouth and the buccal mucosa are treated with radiation therapy or surgery with local control rates of up to 90 per cent. Moderately advanced and advanced cancers of the lip can be controlled effectively by surgery or radiation therapy or a combination of these. Moderately advanced lesions of the retromolar

trigone without evidence of spread to cervical lymph nodes are usually curable, with local control rates of up to 90 per cent. Moderately advanced lesions of the hard palate, upper gingiva and buccal mucosa have a local control rate of up to 80 per cent. In the absence of clinical evidence of spread to cervical lymph nodes, moderately advanced lesions of the floor of the mouth and anterior tongue are generally curable, with survival rates of up to 70 per cent and 65 per cent, respectively. However, for many intraoral cancers, especially those with cervical lymph node metastases, the five year survival rate is less than 50 per cent. The causes of intra-oral SCC (oral cancer) The current hypothesis in oral carcinogenesis is that there is an accumulation of genetic mutations in oral epithelial cells, with tobacco and betel quid mutagens (possibly facilitated by alcohol) identified as aetiological agents. It is estimated that 75 per cent of all intra-oral cancers in Western countries can be attributed to smoking and alcohol. 3 However, only a small proportion of individuals who use tobacco, alcohol or betel quid develop oral cancer and there is an emerging population of oral cancer patients

Table 4. Oral cancer incidence (and mortality) in Australia in 1994 2*

Cancer location ICD9-140 (lip) ICD9-141 (tongue) ICD9-142 (major salivary glands) ICD9-143 (gum) ICD9-144 (floor of mouth) ICD9-145 (other mouth) Males Females Total M:I ratio

668 (8) 255 (6) 923 (14) 0.015 235(120) 135(53) 370(173) 0.468 116 (41) 63(21) 179 (62) 0.364 26 (7) 123 (45) 131 (31) 19 (7) 45 (14) 0.311 43(14) 166 (59) 0.355 92(26) 223 (57) 0.256

National Cancer Institute. CancerNet. Lip and oral cavity cancer. URL:http:// cancernet.nci.nih.gov/clinpdq/soa/Lip_and_oral_cavity_cancer_Physician.html. Accessed June 1999. 148

*The anatomical location of malignancies is coded by the World Health Organization.1 The mortality to incidence (M:I) ratio is calculated by dividing the total number of deaths by the total incidence.
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Fig.1. Keratotic plaque on the mid-buccal mucosa in a long-term cigarette user.The surface irregularity and fissuring make this type of opaque keratosis difficult to clinically assess with confidence and biopsy is indicated.This lesion showed an epithelial hyperplasia with keratosis. Fig. 2. Typical velvet appearance of a speckled leukoplakia/erythroplakia.These lesions are frequently dysplastic and always require histology. This lesion showed a moderate epithelial dysplasia. Fig. 3. Discrete erythroplakia on the soft palate in a male cigarette user. The lesion was asymptomatic and without textural change within the tissues. Biopsy showed a carcinoma in situ. The patient declined any treatment and died from disseminated malignancy several years after initial presentation. Fig.4. Non-homogeneous commissural keratotic plaque showing surface irregularity, erythema and surface desquamation.Such lesions require histology but their clinical presentation may be improved by preliminary use of an antifungal as they frequently have a secondary candidal component. Biopsy showed an epithelial hyperplasia with moderate dysplasia.

who lack exposure to these agents. Infection of oral keratinocytes with human papillomavirus (HPV) may be involved in the development of oral cancer in some patients. A role for HPV in oral cancer is supported by findings of HPV in tumour tissue and by studies showing that HPV immortalizes oral keratinocytes in vitro.4,5 HPV-immortalized oral keratinocytes become tumorigenic in mice following exposure to tobaccorelated chemicals in vitro .6 The E6 and E7 genes of HPV16 and HPV18 encode proteins that are involved in the direct destruction of the p53 and retinoblastoma (pRb) growth-regulatory proteins, respectively.4 In contrast, mutagens in tobacco, alcohol and betel quid are thought to damage important growth-regulatory genes in oral kerat i n o cy t e s , resulting in cancer formation. Chronic infection of oral keratinocytes with Candida albicans may have a role in oral carcinogenesis. Candida albicans stimulates epithelial cell proliferation in vitro and oral lesions of chronic hyperplastic candidosis (candidal leukoplakia) show epithelial dysplasia and may undergo malignant
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transformation.7 F u rt h e rm o r e , C. albicans was shown in rats to be an effective promoter of 4-nitroquinoline-1-oxide (4NQO)-initiated oral mucosal squamous cell carcinoma. 8 Oral cancer and epithelial dysplasia It is unknown if all oral cancers are preceded by histological epithelial dysplasia.This is mainly because most oral cancers (at least in Western societies) are not preceded by a visual lesion and therefore very few patients have a precancer biopsy at the cancer site. Epidemiological data suggests that a proportion of many oral mucosal lesions including idiopathic white patch (leukoplakia) (Fig. 1), speckled leukoplakia (Fig. 2), erythroplakia (Fig. 3), chronic hyperplastic candidosis (Fig. 4), oral submucous fibrosis, discoid lupus erythematosus and dyskeratosis congenita undergo malignant transformation. However, biopsy sections of these lesions show marked variability in the presence of epithelial dysplasia.7 Only a few studies have followed dysplastic oral
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lesions histologically over a period of time.In a study of 45 patients with dysplastic oral lesions followed for up to eight years, 11 per cent of lesions became malignant while 30 per cent regressed spontaneously.9 It seems that oral cancers are preceded by histological epithelial dysplasia (which may or may not be apparent clinically), although not all dysplastic oral lesions progress to malignancy. At present, it is not possible to predict which dysplastic oral lesions will proceed to malignancy and which will regress.10 A recent molecular study found identical genetic changes in dysplastic oral lesions and oral cancers in the same patients, suggesting that the progeny of cells in a dysplastic oral lesion may, with further modification, ultimately form a malignant lesion.11 This is the first evidence that a clone of oral keratinocytes proceeds through stages of transformation from normal, to dysplastic, to malignant. The finding suggests that oral keratinocytes suffer genetic damage at each stage boundary until the accumulated genetic damage is sufficient for autonomous proliferation and tumour formation. Oral cancer and cell division An obvious feature of all oral cancers is excessive proliferation of oral keratinocytes. Initially, keratinocyte proliferation may be confined to the epithelial compartment resulting in a thickened and disorganized epithelium. Individual keratinocytes show nuclear hyperchromatism (dark staining), nuclear pleomorphism (abnormal shape), enlarged nucleoli, increased nuclear:cytoplasmic ratio, increased mitoses, suprabasal mitoses, abnormal mitoses and multinucleation. There may be loss of polarity of the basal cells, drop-shaped rete processes, irregular epithelial stratification and reduced cellular cohesion. The intra-epithelial lesion is termed epithelial dysplasia (graded as mild, moderate or severe) or carcinoma in situ , depending on extent. Eventually, the proliferating keratinocytes break through the epithelial basement membrane (see The spread of oral cancer below). Malignant epithelial masses then expand through the underlying connective tissue and invade lymph and blood vessels resulting in distant spread. In this context, oral cancer is a lesion characterized by dysregulated division of oral keratinocytes. Knowledge of normal DNA replication and keratinocyte division is the key to understanding abnormal cell division in oral cancer. Normally, oral keratinocyte division is stimulated by epidermal growth factor (EGF) binding the epidermal growth factor receptor (EGFr) on the surface of basal keratinocytes (Fig. 5).12 EGF receptor-ligand interaction activates the ras protein on the cytoplasmic side of the EGFr. Active ras activates the Raf protein and subsequently the other cytoplasmic kinases (MEK, MAPK) in a
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cascade-like manner (Fig. 5). MAPK is mitogen activated protein (MAP) kinase, MEK is MAP kinase kinaseand Raf is MAP kinase kinase kinase. The kinase cascade transmits the growth signal from the cell membrane to the nucleus where the level of c-myc protein rises sharply (Fig. 5). The c-myc protein binds DNA and stimulates the transcription of cyclin D which binds and activates cyclin-dependent kinase (CDK). Active CDK catalyses the phosphorylation of the retinoblastoma tumour suppressor protein (pRb). Phosphorylated pRb releases the E2F transcription factors which are required for the transcription of DNA replication proteins (Table 5). DNA replication proceeds, followed closely by cell division (Fig. 5). Cyclin D and most of the DNA replication proteins are degraded and must be newly transcribed with each round of cell division. Many of the proteins which transmit the growth signal from the cell membrane to the nucleus are encoded by oncogenes. As discussed below, oncogene mutation may stimulate excessive keratinocyte proliferation in oral cancer. The genetic basis of oral cancer Evidence suggests that oral cancer results from genetic damage.There is increased risk of oral cancer associated with exposure to genetic mutagens in tobacco, alcohol and betel quid. Gene mutations have been detected in oral SCC in chromosomes 3p, 4q, 6p, 8p, 9p, 11q, 13q [retinoblastoma (Rb) tumour suppressor gene], 14q, 17p (p53 tumour suppressor gene), 18q [deleted in colon cancer (DCC) tumour suppressor gene] and 21q.11,13 The genetic hypothesis predicts a role for hyperactive oncogenes (growth promoting genes) in oral carcinogenesis. Oncogenes encode many of the signal-transmitting proteins (for example, EGFr, ras, cytoplasmic kinases, c-myc) via which cells respond to external growth signals (Fig. 5). Normal cells, with normal oncogenes, will not commit themselves to another

. Enzymes required in nucleotide synthesis . Initiator proteins bind at replication origin . DNA helicase binds initiator proteins and opens the double helix . Single strand DNA binding proteins (helix destabilizing proteins) stabilizes unwound single strand DNA . DNA polymerase polymerizes deoxyribonucleoside triphosphates on a single strand DNA template . (5-to-3) PCNA (proliferating cell nuclear antigen) tethers DNA to the DNA template . polymerase DNA primase makes RNA primers for lagging strand replication . DNA ligase joins Okazaki fragments during lagging strand replication . Proofreading exonuclease provides base-paired terminus that primes DNA synthesis . DNA topoisomerase transient nick in DNA to allow free rotation
during replication *Most of these proteins are enzymes which are newly transcribed with each round of cell division. Many of these proteins are transcribed by the E2F family of transcription factors (Fig.5).
Australian Dental Journal 1999;44:3.

Table 5. Proteins required for DNA replication*

round of DNA replication and cell division without stimulation from such external signals. However, with oncogene mutation, the mutant oncoprotein may send a growth-stimulatory signal to the nucleus, regardless of events taking place in the cells surroundings. The subsequent autonomous proliferation of mutant oncogene-bearing cells results in tumour formation. As discussed, the ras oncoprotein lies on the internal aspect of the membrane-bound EGFr and is involved in the transmission of the EGF growthstimulatory signal to the nucleus (Fig. 5). In the inactive state, ras binds guanosine diphosphate (GDP).When cells are stimulated by EGF, inactive ras become activated by exchanging GDP for guanosine triphosphate (GTP). Active ras activates the Raf protein and subsequently the other cytoplasmic kinases (MEK, MAPK) in a cascade-like manner (Fig. 5). After activating Raf, active ras returns to the inactive state due to its intrinsic guanosine triphosphatase (GTPase) activity. GTPase hydrolyses GTP to GDP, thereby releasing a phosphate group and returning the ras protein to its inactive GDPbound state. The intrinsic GTPase activity of activated ras is amplified by binding of GTPaseactivating proteins (GAPs). Under normal circumstances, this mechanism ensures that ras is active for only a brief period of time following EGF stimulation and that cell proliferation is tightly regulated by EGF. However, the control of ras activity changes dramatically with mutation of the ras oncogene. Mutant ras protein can bind GAPs, however GTPase activity is not amplified. Hence, mutant ras is trapped in its active GTP-bound form which continues to activate Raf, thus sending a proliferation signal to the nucleus even in the absence of binding between EGF and its receptor on the cell surface.14 In addition, hyperactive ras may stimulate the transcription of bcl-2 which blocks caspase 3 activation and hence blocks apoptotic cell death (discussed below). Many studies have shown ras oncogene mutation and ras oncoprotein over-expression in oral SCC.12,15 Furthermore, oral premalignant lesions and oral cancer may be associated with upregulated EGF receptor expression.16 Oral cancer may, therefore, result from mutation of an oncogene in the EGF pathway. The mutant oncoprotein sends a continuous growth signal to the nucleus resulting in continuous oral keratinocyte proliferation and tumour development. Under normal circumstances, the p53 tumour suppressor protein detects DNA damage (such as ras oncogene mutation) and halts progression through the cell cycle. Following DNA damage, there is an increase in the level of p53 which, in turn, stimulates the transcription of p21 (Fig. 5). The p21 tumour suppressor protein is a CDK inhibitor and blocks
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F i g .5 . Normal oral keratinocyte division is stimulated by epidermal growth factor (EGF) binding the EGF receptor (EGFr) which activates ras. Active ras triggers the kinase cascade (Raf, MEK, MAPK) resulting in increased levels of c-myc in the nucleus. c-myc stimulates the transcription of cyclin D which activates cyclin-dependent kinase (CDK). Active CDK catalyzes the phosphorylation of the retinoblastoma tumour suppressor protein (pRb). Phosphorylated pRb releases the E2F transcription factors which are required for the transcription of DNA replication proteins including proliferating cell nuclear antigen (PCNA). DNA replication proceeds, followed closely by cell division. Cyclin D and most of the DNA replication proteins are degraded and must be newly transcribed with each round of cell division. DNA damage in oral keratinocytes is detected by p53.As a result, there is an increase in the level of p53 which stimulates the transcription of p21, a CDK inhibitor which blocks the phosphorylation of pRb. p21 also binds and deactivates PCNA. p53 stimulates the transcription of Bax which blocks the activity of bcl-2. Caspase 3 activity is therefore unchecked and apoptotic cell death proceeds. The dotted lines represent the apoptotic cell death pathway following DNA damage.

the phosphorylation of pRb, thereby blocking the release of E2F transcription factors and blocking DNA replication.The p21 tumour suppressor protein also binds and deactivates proliferating cell nuclear antigen (PCNA) (Fig. 5). PCNA is a toroidalshaped protein that encircles and slides along DNA. PCNA tethers the DNA polymerase catalytic unit to the DNA template and is therefore essential for DNA replication. Binding of p21 to PCNA inhibits the interaction between PCNA and DNA polymerase, thereby blocking DNA replication. Hence p53, acting via p21, puts the brakes on DNA replication and cell division in cells with damaged DNA. In some instances, p53 will trigger apoptosis (programmed cell
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death) in cells with damaged DNA. p53 stimulates the transcription of Bax which blocks the activity of bcl-2 (Fig.5). bcl-2 normally blocks the activation of caspase 3, a central mediator of the apoptosis cell death pathway. When bcl-2 activity is blocked by Bax, caspase 3 activity is unchecked and apoptotic cell death proceeds (Fig. 5). p53 also represses the transcription of bcl-2 which further contributes to caspase 3 activity and apoptosis in cells with damaged DNA. However, if the p53 gene is mutated, the protection offered by the p53 tumour suppressor protein against DNA damage (for example, ras oncogene mutation) is lost. Both alleles of the tumour suppressor gene must be mutated for the tumour suppressor protein to become non-functional. A cell which is heterozygous for a tumour suppressor gene has one normal allele and one mutant allele and the cell is clinically normal. If a cell becomes homozygous for the mutant allele, that is loses heterozygosity, cancer may develop. In familial cases of retinoblastoma, children inherit one mutant allele of the Rb tumour suppressor gene and the other allele is normal. Only one somatic mutation is required to inactivate the single normal allele of the Rb gene and for retinoblastoma to develop. In sporadic cases, both normal alleles of the Rb tumour suppressor gene in a single retinoblast are lost by somatic mutation resulting in retinoblastoma. In the Li-Fraumeni cancer susceptibility syndrome, children inherit one mutant allele of the p53 tumour suppressor gene and the other allele is normal. Again, only one somatic mutation is required to inactivate the single normal allele of the p53 gene and patients with this syndrome are at high risk of developing carcinomas, sarcomas, lymphomas and brain tumours. Most oral cancers are sporadic and therefore both normal alleles of the p53 tumour suppressor gene in a single oral keratinocyte are lost by somatic mutation. In this context, the tumour suppressor genes are recessive, as both alleles must be damaged for transformation to occur. In contrast, the oncogenes (for example, ras) are dominant, as mutation of only one allele results in transformation. Mutation of the p53 tumour suppressor gene is the most common genetic lesion in human neoplasia and p53 is mutated in up to 80 per cent of oral cancers.17 In oral SCC, p53 mutation correlates with a history of heavy smoking and is associated with increased epithelial cell proliferation. Mutation of p53 may precede18 or accompany19 the transition from oral pre-cancer to cancer. A common p53 mutation in many tumours is deletion of one allele accompanied by a mutation in the central DNA-binding domain of the remaining allele. Consequently, the mutant p53 protein is unable to stimulate p21 or Bax transcription and cells with damaged DNA (for
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example, mutated ras oncogene) continue to divide, passing on their gene mutations to subsequent generations. By way of a simple example, oral cancer may result from an activating mutation of the ras oncogene and a simultaneous deactivating mutation of the p53 tumour suppressor gene in an oral keratinocyte. In this scenario, oral cancer would evolve from an oral keratinocyte that has received three genetic hits from mutagens in tobacco, alcohol, betel quid or some other source. The first hit would inactivate one allele of the p53 tumour suppressor gene. The second hit would inactivate the other allele of the p53 tumour suppressor gene. The third hit would activate the ras oncogene.The order of these hits is probably important, as ras oncogene mutation in the presence of at least one functional allele of p53 would be expected to result in cell cycle arrest and apoptosis. As discussed, oral cancer may result from mutation in other oncogenes in the EGFr/ras/kinase cascade/c-myc signalling pathway and Califano et al. identified cumulative tumour suppressor gene mutations which correlated with the stages of carcinogenesis from normal mucosa, to squamous hyperplasia (9p), to dysplasia (3p, 17p) to carcinoma in situ (11q, 13q, 14q) to invasive carcinoma (6p, 8 and 4q). 13 Field cancerization Some oral cancer patients develop SCC over a broad area of the oral mucosa, with multiple lesions arising simultaneously or over a period of time.The high incidence of second primary cancers in patients with oral SCC was first reported by Slaughter et al. in 1953.20 The authors proposed the term field cancerization to describe this phenomenon and subsequent reports have confirmed their observations.21 Approximately 2-3 per cent of oral cancer patients develop a second primary cancer each year after removal of the primary tumour and 90 per cent of recurrences manifest within two years of initial treatment.With advances in therapy, more patients survive initial tumours. Hence, the incidence of second primary oral cancers is expected to rise.22 U n f o rt u n at e l y, tumour recurrence or a second primary tumour has a significant adverse effect on survival of oral cancer. Hence, the identification of a predictive marker for second primary oral cancers would have significant prognostic and patient management implications. Until that time, the rising incidence of second primary tumours indicates that prevention should be the primary objective. The mechanisms of field cancerization are unknown, although three basic hypotheses were proposed recently by Ogden.22 Firstly, field changes (molecular changes throughout the oral mucosa of oral cancer patients) may predispose to the development of multiple primary cancers. In this scenario, a
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large region of the oral mucosa may be exposed to the aetiological agent(s) which causes independent transformation of multiple epithelial cells at separate sites. A single aetiological agent acting at different sites would cause multiple separate cancers with identical genetic defects, each arising as a separate clone within the oral mucosa. Subsequent genetic modification (due to spontaneous mutation or continued exposure to exogenous mutagens) may render the separate clones genetically distinct. Different aetiological agents acting at different sites would cause multiple separate cancers with different genetic defects, each arising as a separate clone within the oral mucosa. 22 Secondly, the aetiological agent(s) may transform a single oral epithelial cell. The expanding clone of cancer cells may spread through the oral mucosa via local tissue spread, regional blood vessels, seeding via the saliva into a mucosal erosion or seeding due to the trauma of surgery. This would give rise to geographically distinct but genetically identical cancers. Again, subsequent genetic modification (due to spontaneous mutation or continued exposure to exogenous mutagens) may mask the clonal origin of tumours at different sites.22 Studies have identified changes in the histologically normal epithelium adjacent to oral cancers including reduced cytoplasmic area, alterations in keratin expression, upregulated EGF receptor expression and p53 mutation. These changes suggest that genetically altered epithelial cells migrate from the pre-neoplastic (dysplastic) lesion into the surrounding normal oral epithelium.The clinical significance of p53 mutation within the normal oral epithelium of oral cancer patients is unclear. Some reports suggest an association with the development of second primar y cancers23 while others find no such association.24 Recent molecular studies have shown that oral cancer is a clonal proliferation of neoplastic keratinocytes (that is, oral cancers arise from a single genetically altered cell) and that multiple primary tumours result from the migration of clonally-related preneoplastic cells through the oral epithelium.13,25 Thirdly, a tumour may have a paracrine effect on the adjacent oral mucosa. Of great recent interest is that tumours have been found to secrete tumour inhibitory factors including inhibitors of neovascularization. The angiogenesis inhibitor Endostatin was originally isolated from a murine haemangioendothelioma. Endostatin is a potent inhibitor of endothelial cell growth and its effect on human solid tumours (lung cancer, lymphoma, breast cancer, colon cancer, prostate cancer) is being examined in clinical trials currently at the University of

Wisconsin, Madison, USA and the University of Texas, M. D. Anderson Cancer Center, Houston, USA. Removal of the primary tumour would remove these inhibitors of cancer development and hence promote second primary tumour formation. Alternatively, tumours may secrete promoters of apoptosis. Removal of the primary tumour would reduce the level of apoptosis in adjacent tissue and hence promote second primary tumour formation.22 The spread of oral cancer As discussed, oral keratinocyte proliferation is confined initially to the epithelial compartment. Eventually, the proliferating keratinocytes break through the epithelial basement membrane and cell masses expand through the underlying connective tissue and invade lymph and blood vessels, resulting in distant spread. Conventional transmission electron microscopy shows that the epithelial basement membrane consists of a lamina lucida which underlies the basal keratinocyte plasma membrane and a lamina densa located between the lamina lucida and the underlying connective tissue. The basement membrane is composed of laminins 1 and 5, type IV collagen and heparan sulphate proteoglycan (HSPG). Hemidesmosomes anchor the basal keratinocytes to the basement membrane and underlying connective tissue. Hemidesmosomes consist of keratinocyte cytoplasmic filaments which are linked via 64 integrin to external anchoring filaments. Laminin 5, a component of the anchoring filaments, may serve as the ligand for 64 integrin. Anchoring filaments penetrate the basement membrane and join anchoring fibrils in the connective tissue. Anchoring fibrils are composed of type VII collagen. Thus, the epithelial basement membrane is a formidable proteinaceous barrier to tumour cell migration. Tumour cells penetrate the basement membrane barrier and spread through the underlying connective tissue with the help of matrix protein degrading enzymes called matrix metalloproteinases (MMPs). MMPs are produced by the tumour cells themselves or the tumour may stimulate cells in the surrounding tissue (stroma) to produce MMPs.26 The MMPs are a family of zinc-containing endo-proteinases and the MMP gene family encodes more than 20 different MMPs. MMPs are grouped by their substrate specificity: 1. The collagenases (MMP-1 and MMP-8) cleave collagen I, II and III preferentially. 2. The stromelysins (MMP-3, MMP-10 and MMP-11) cleave laminin. 3. The gelatinases (MMP-2 and MMP-9) cleave type IV collagen. Many other matrix proteins are potential substrates of MMPs including fibronectin, elastin, decorin,
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vitronectin and proteoglycans. MMPs are secreted in an inactive form and activated subsequently by other active MMPs, membrane type MMPs (MTMMPs) or mast cell chymase. The activation of MMPs is regulated partially by tissue inhibitors of metalloproteinases (TIMPs).The principal function of MMPs is the proteolytic degradation of connective tissue matrix proteins. MMPs are involved in normal growth and development, tissue remodelling and angiogenesis. MMPs are also involved in tumour invasion and metastasis.26 MMP-1, MMP-2,MMP-3 and MMP-9 are highly expressed in oral cancers and the surrounding stroma (especially at the invading front of the tumour) and high levels of MMP expression may indicate a poor prognosis.27 Triggering of the v6 integrin, an adhesion molecule expressed by oral cancer cells, upregulates production of MMP-2 and MMP-9 which cleave basement membrane proteins and thus facilitate oral cancer penetration of the epithelial basement membrane.28 MMPs also have a role in tumour angiogenesis which is essential for tumour growth and spread.26 In this context, recent studies have identified vascular endothelial growth factor (VEGF) in tumour cells and VEGF receptor expression by endothelial cells in oral cancer, both of which were associated with lymph node metastasis.29 Oral cancer prevention Despite advances in oral cancer therapy, the survival rate for oral cancer is showing no signs of improvement. 30 Therefore, emphasis should be placed on oral cancer prevention. Epidemiological data indicate that exposure to tobacco, alcohol and betel quid is associated with an increased risk of oral SCC. Primary prevention involves reducing exposure to these carcinogens and has been shown to be effective in reducing the incidence of oral cancer. Secondary prevention involves screening for the early detection of oral cancer. Although easily detected and often cured in its early stages,most oral cancers are moderately advanced and have spread to regional lymph nodes at the time of diagnosis. It seems, therefore, that screening for oral premalignant lesions and early oral cancers would decrease oral cancer morbidity and mortality. However, the National Cancer Institute (NCI) in the USA does not recommend screening for oral cancer and points out that there is insufficient evidence to establish that screening would decrease oral cancer mortality. The authors are currently seeking funds to undertake a pilot oral cancer screening program in Australia. Data from this investigation will address directly the

National Cancer Institute. CancerNet. Screening for oral cancer. URL:http:// cancernet.nci.nih.gov/clinpdq/screening/Screening_for_oral_cancer_Physician. html.Accessed June 1999. 154

NCI concerns regarding the efficacy of oral screening. Due to the cost of population screening, it is planned to initially target high-risk groups including smokers and heavy drinkers. However, experience has shown that the identification and screening of high-risk individuals is difficult and smokers are often reluctant to accept an invitation for examination.31 Oral cancer screening can take many forms. Clinical and histological examination allows the early detection of oral premalignant and malignant lesions. Oral cancer occurs in a region of the body that is generally accessible to physical examination by the patient, the dentist and the physician. Screening can be made more efficient by inspecting the high-risk sites where 90 per cent of all oral squamous cell cancers arise; the floor of the mouth, the ventrolateral aspect of the tongue and the soft palate. The authors recommend that dentists perform an annual visual oral cancer examination on all of their patients and obtain a specialist opinion for suspicious oral lesions, including idiopathic white patch (leukoplakia) (Fig. 1), speckled leukoplakia (Fig. 2) and erythroplakia (Fig. 3). Unfortunately, in Western societies, most oral cancers are not preceded by a visual lesion, thus limiting the usefulness of visual oral examination.7 More frequent oral cancer examinations are recommended for treated oral cancer patients to monitor the development of second primary tumours.20,21 Family members of patients with oral cancer are also at higher risk and therefore should be examined more frequently.32 Clearly, clinical examination by a dentist is inefficient for population-based oral screening. Exfoliative cytology can detect early oral cancer and can be performed by dentally-untrained personnel. Exfoliative cytology is rapid and relatively non-invasive and therefore may be useful in population-based oral cancer screening programs. However, as with cervical cytology, oral cytology can give false negative findings due to inadequate sampling of lesions and the subjective assessment of smears. Recent studies have shown that automated, quantitative smear assessment enhances the diagnostic reliability of oral exfoliative cytology.33 Whatever screening method is used, a positive screening result must be confirmed by definitive investigation including biopsy. The future promise for oral exfoliative cytology is to identify early markers of oral cancer and markers of oral cancer susceptibility, including cytokeratin and heat shock protein expression, oncogenic viruses and mutations in oncogenes and tumour suppressor genes.34,35 Individuals with a genetic susceptibility to oral cancer thus may be identified and preventive measures instigated, including the elimination of exogenous carcinogens and regular monitoring. In addition, the risk of oral cancer may be reduced with dietary modification, including increased consumption of
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fresh fruit, fresh vegetables (particularly carrots, tomatoes, capsicum and green leaf) and possibly the micro-nutrients vitamin C, vitamin A and the vitamin A precursor beta-carotene, although convincing evidence that individual micro-nutrients are protective is lacking. Reducing total calories, fat, butter, eggs and starchy foods may further reduce the risk of oral cancer.3 As discussed, a proportion of some benign oral mucosal lesions undergo malignant transformation. As a form of oral cancer prevention, these lesions should be treated with the aim of preventing malignant change.10 The clinical appearance of an oral lesion, especially a homogeneous white patch, is unreliable as a predictor of malignant transformation. Oral white lesions in women are more likely to undergo malignant transformation than those in men. Other risk factors for malignant transformation of oral white lesions include a long duration,involvement of the floor of mouth or tongue, a non-homogeneous appearance, the presence of C. albicans, a previous cancer history, a family history of cancer and ongoing exposure to aetiological agents (tobacco, alcohol, betel quid). 36 The presence of epithelial dysplasia in a biopsy section is suggestive of malignant potential, although a percentage of dysplastic lesions regress spontaneously.7 F u rt h e rm o r e , a normal biopsy result must be viewed with caution as the degree of dysplasia often varies throughout such lesions. In future, the identification of oncogene and tumour suppressor gene mutations in biopsy specimens may give a clearer indication of the likely behaviour of suspicious oral lesions. The choice of treatment for a suspected premalignant oral lesion depends on the history of the lesion, the clinical appearance, the extent of the lesion, the degree of epithelial dysplasia, the risks and benefits of various therapies and the expected level of patient compliance.10,36 The current recommendation is: 1. Biopsy with or without vital tissue staining (toluidine blue or Lugols iodine) 2. Elimination of risk factors (tobacco, alcohol, betel quid, C. albicans). 3. Anti-inflammatory and antimycotic drugs. 4. Surgical resection (scalpel or laser) of persistent lesions with histology.10 The treatment of suspected premalignant oral lesions with retinoids (13-cis-retinoic acid, isotretinoin, fenretinide), beta-carotene, vitamin E, bleomycin and alpha-tocopherol is under evaluation.36 The main problems with these agents is their toxicity and the recurrence of lesions following withdrawal of treatment.10 No treatment guarantees elimination of malignant potential and therefore careful follow-up is required.36
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Future therapies for oral cancer Based on our understanding of oral cancer genetics, growth and spread, many new oral cancer therapies are possible. These include monoclonal antibodies against the EGF receptor, gene therapy to deactivate oncogenes and reactivate tumour suppressor genes and inhibition of MMPs to block tumour spread and metastasis. A prominent area in cancer research is the study of a group of compounds called angiogenesis inhibitors which block the development of new blood vessels. Solid tumours cannot grow beyond 1-2 mm in diameter without inducing the formation of new blood vessels to supply nutrients and oxygen to the tumour. Blocking the development of new blood vessels starves the tumour of nutrients and oxygen, thereby inhibiting tumour growth and spread to other parts of the body. Drug resistance is a major problem with traditional chemotherapeutic agents. Most cancer cells are genetically unstable and often produce drug resistant cells. In contrast, the angiogenesis inhibitors target normal endothelial cells which are genetically stable. Hence, resistance to angiogenesis inhibitors is less likely to develop and data from long-term animal studies and preliminary clinical trials has shown this to be the case. Currently, four anti-angiogenesis strategies are being investigated in clinical trials involving more than 30 different angiogenesis inhibitors: 1. Block the ability of the endothelial cells to break down the surrounding matrix using, for example, Marimastat which is a synthetic MMP inhibitor. 2. Inhibit endothelial cells directly using, for example, Endostatin which inhibits endothelial cell growth. 3. Block factors that stimulate angiogenesis, for example, SU5416** which blocks VEGF receptor signalling. 4. Block the action of v3 integrin,a molecule on the endothelial cell surface which enhances endothelial cell survival and promotes angiogenesis using, for example,Vitaxin which is an anti-integrin antibody. Further information about clinical trials of angiogenesis inhibitors is available from the National Cancer Institute, USA. Conclusion As discussed, the survival rate for oral cancer is showing no signs of improvement. Therefore, until more effective oral cancer therapies are available, the

British Biotech,Annapolis,MD, USA. **Sugen,South San Francisco, CA,USA. Ixys,La Jolla,CA,USA. National Cancer Institute. CancerNet.Anti-angiogenesis information.URL: h t t p : / / c a n c e rt ri a l s. n c i . n i h . g ov / N C I _ C A N C E R _ T R I A L S / z o n e s / P r e s s I n f o / Angio.Accesssed June 1999. 155

emphasis should be placed on primary prevention (reducing exposure to causative agents) and secondary prevention (early detection and treatment). Acknowledgements The authors original work has been funded in part by the National Health and Medical Research Council (Australia) and the Australian Dental Research Foundation Inc. References
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Address for correspondence/reprints: Dr Philip Sugerman, Oral Biology and Pathology, Department of Dentistr y, Floor 5, Physiology Building, The University of Queensland, St Lucia, Queensland 4072.
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